1. Study Guide/Outline—Bacterial Gene Regulation Bacterial Gene Regulation What is an operon? How is it different from a eukaryotic gene? In the lac operon, what cellular or environmental conditions must exist in order for the (WT) lac operon to express its genes? How do these environmental conditions positively or negatively regulate the operon? What are the different parts, and their functions, of the operon? How do mutations in “upstream” parts of the operon (promoter, operator, coding genes) affect the “downstream” areas of the operon? How do missense and nonsense mutations have different results? The lacI gene is not part of the Lac Operon. How is the lac I gene involved with the Lac operon? What kinds of mutations are cis-dominant? Trans-dominant? Constitutive ON? Constitutive-OFF? How can a bacteria be a partial diploid? How does being diploid for the LacI gene create complexities in the regulation of the Lac Operon?

2. Brooker Fig 16.3b Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display H H H H OH HO CH 2 OH O HOH Galactose H H H OH H CH 2 OH O HOH H H H H HO CH 2 OH O HOH H H H O H CH 2 OH O O HOH H H H H HO CH 2 OH O HOH H H+H+ H+H+ H H H CH 2 O O HOH Glucose +  galactosidase β  galactosidase side reaction Allolactose Lactose Cytoplasm Lactose Lactose permease Functions of lactose permease and  -galactosidase

3. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Brooker Fig 16.8 Promoter CAP site Operator (b) No lactose or glucose (high cAMP) cAMP Transcription is very low due to the binding of the repressor. Repressor CAP cAMP CAP Promoter CAP site Operator Repressor (inactive) High rate of transcription Allolactose Binding of RNA polymerase to promoter is enhanced by CAP binding. (a) Lactose, no glucose (high cAMP) Positive control—Catabolite Activator Protein (CAP) turns on Lac Operon High rate of transcription But negative control Must be removed before positive control will result in transcription

4. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Brooker, Fig 16.8 Allolactose Promoter CAP site Operator Repressor (inactive) (Inactive) (c) Lactose and glucose (low cAMP) Promoter CAP site Operator (d) Glucose, no lactose (low cAMP) Transcription rate is low due to the lack of CAP binding. CAP Transcription is very low due to the lack of CAP binding and the binding of the repressor. CAP In absence of cAMP, transcription is very low (or hardly at all)

5. lac repressor binds to the operator and inhibits transcription. lac regulatory gene lac operon mRNA lacI lacP lacO lacZlacY lacA lac repressor (active) (a) No lactose in the environment Figure 16.4 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Constitutive expression of lacI Promoter Operator RNA pol cannot access the promoter when repressor bound to operator

6. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Figure 16.4 RNA polymerase mRNA lacI lacP lacO lacZlacY lacA Allolactose Transcription (b) Lactose present  galactosidase Lactose permease Galactoside transacetylase The binding of allolactose causes a conformational change that prevents the lac repressor from binding to the operator site. Conformational change Polycistronic mRNA Lactose causes repressor to fall off Operator Site

7. Figure 16.5a Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Lac repressor Lactose permease  galactosidase Lactose Lac repressor Transacetylase 4. Most proteins involved with lactose utilization are degraded. 1. When lactose becomes available, a small amount of it is taken up and converted to allolactose by β-galactosidase. The allolactose binds to the repressor, causing it to fall off the operator site. 2. lac operon proteins are synthesized. This promotes the efficient metabolism of lactose. 3. The lactose is depleted. Allolactose levels decrease. Allolactose is released from the repressor, allowing it to bind to the operator site. Lac repressor lac operon Induction of Lac Operon

8. Animation Lac Operon http://vcell.ndsu.nodak.edu/animations/

9. Brooker Figure 16.7 4. Incubate the cells long enough to allow lac operon induction. 5. Burst the cells with a sonicator. This allows β-galactosidase to escape from the cells. – Lactose F’ F In mero-zygote strain, the lac I + gene on the F´ factor makes enough repressor to bind to both operator sites (restoring WT phenotype on main chromosome). Lactose is taken up, is converted to allolactose, and removes the repressor. 1234 Lactose 3. + Lactose 4. Z+Z+ I–I– P O Y+Y+ A+A+ Z+Z+ P O Y+Y+ I+I+ Experimental levelConceptual level 1. Grow mutant strain and merozygote strain separately. 2.Divide each strain into two tubes. 3. In one of the two tubes, add lactose. Mutant strain Merozygote strain Merozygote – Lactose Mutant F′ Operon is constitutive-on in Mutant strain because no repressor is made. 1. + Lactose 2. Z+Z+ I–I– P O Y+Y+ A+A+ Z+Z+ I–I– P O Y+Y+ A+A+ Z+Z+ P O Y+Y+ A+A+ I+I+ Z+Z+ I–I– P O Y + A+A+ Z + I – P O Y + A + A+A+ Z+Z+ I–I– P O Y+Y+ A+A+ Z+Z+ P O Y+Y+ I+I+

10. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 6. Add β-o-nitrophenylgalactoside (β-ONPG). This is a colorless compound. β-galactosidase will cleave the compound to produce galactose and o-nitrophenol (O-NP). O-NP has a yellow color. The deeper the yellow color, the more β-galactosidase was produced. 7. Incubate the sonicated cells to allow β-galactosidase time to cleave β-ONPG. 8. Measure the yellow color produced with a spectrophotometer. (See the Appendix for a description of spectrophotometry.)  o-nitrophenyl- galactoside O-NP  -galactosidase Broken cell 1. 1234 NO 2 + 2. + 3. 4. +  ONPG Galactose NO 2 Brooker Figure 16.7, cont

11. Table 16.116 – 34

12. Question Will a loss-of-function mutation in P lac (promoter sequence) be cis-dominant or trans-dominant?

13. Lactose status (assume absence of Glucose) GenotypePromoter Seq RepressorOperator Seq Lac Z Lac Y Lac A Type of mutation (e.g. cis- dominant, consititutive ON) AbsentWT + Active Bound No Expression none PresentWT + InactivatedOpenWT B-Gal WT Permease WT Transacet. none PresentLac Y miss PresentLac Z Nons PresentP Lac(-) AbsentLac O c PresentLac O c

14. Lactose status (assume absence of Glucose) Genotype Promoter Seq Repressor Operator Seq Lac ZLacY Lac A Type of mutation (e.g. cis- dominant) AbsentLac I (-) Absent F’-Lac I (+) Lac I (-) Absent F’-LacO c Lac O+ Present F’-LacO c Lac O+

15. Go over lecture outline at end of lecture